The mechanism of membrane associated transport functions is poorly understood and the mechanism of membrane assembly and biogenesis is essentially unknown. In this study, a novel mutant (Yf) of the lac permease of Escherichia coli is used as a probe to study the mechanisms of biogenesis and transport. This mutant is defective both in transport function and in its assembly of the lac permease as an active membrane component. The assembly defect of the Yf permease is dominant over the assembly of the wild-type permease. A model explaining the assembly defect of the Yf permease in terms of a stable permease precursor which accumulates in the cytoplasm is consistent with preliminary fractionation studies. The mechanism of biogenesis and assembly of the lac permease will be studied using the Yf mutant as a probe in double-label, pulse-chase experiments. A purification scheme will be developed and the purified precursor and permease will be studied in order to determine the post-translational modifications required leading to activity. The Yf mutant will be exploited in a genetic study in order to determine the number of lac operon cistrons involved in permease function and assembly. Finally, the Yf mutant will be used to study the mechanism of transport and to determine whether or not the functional permease is a multimeric protein.